Engineered opsonin for pathogen detection and treatment

ABSTRACT

The present invention provides for engineered molecular opsonins that may be used to bind biological pathogens or identify subclasses or specific pathogen species for use in devices and systems for treatment and diagnosis of patients with infectious diseases, blood-borne infections or sepsis. An aspect of the invention provides for mannose-binding lectin (MBL), which is an abundant natural serum protein that is part of the innate immune system. The ability of this protein lectin to bind to surface molecules on virtually all classes of biopathogens (viruses, bacteria, fungi, protozoans) make engineered forms of MBL extremely useful in diagnosing and treating infectious diseases and sepsis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/831,480, filed Aug. 20, 2015, which is a Continuation of U.S.application Ser. No. 13/574,191, filed Oct. 23, 2012, now U.S. Pat. No.9,150,631, which is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application No. PCT/US2011/021603, filed Jan. 19, 2011,which designates the U.S., and which claims the benefit of U.S.Provisional Application No. 61/296,222 filed Jan. 19, 2010, the contentsof each of which are incorporated fully herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 20, 2015, isnamed 20150820_Sequence_Listing_002806-078071-C.txt and is 7,084 bytesin size.

FIELD OF THE INVENTION

The present invention relates to molecular immunology, microbialpathogens, and systems for detecting and/or removing pathogens influids, including bodily fluids such as blood. More specifically, forexample, the present invention provides for an engineered molecularopsonin that may be used to bind biological pathogens or identifysubclasses or specific pathogen species for use in devices and systemsfor treatment and diagnosis of patients with infectious diseases,blood-borne infections, or sepsis.

BACKGROUND

In the U.S., sepsis is the second-leading cause of death in non-coronaryICU patients, and the tenth-most-common cause of death overall. Sepsisis a serious medical condition that is characterized by a whole-bodyinflammatory state (called a systemic inflammatory response syndrome)and the presence of a known or suspected infection. Sepsis typicallyoccurs during bacteremia, viremia or fungemia, and may result frominfections that are caused by pathogens, such as Staphylococcus aureus,that are not typical bloodborne pathogens. Bloodborne pathogens aremicroorganisms that cause disease when transferred from an infectedperson to another person through blood or other potentially infectedbody fluids. The most common diseases include Hepatitis B, HumanImmunodeficiency Virus, malaria, Hepatitis C, and syphilis.

Unfortunately, systemic inflammatory response syndrome may become lifethreatening before an infective agent has been identified by bloodculture. This immunological response causes widespread activation ofacute-phase proteins, affecting the complement system and thecoagulation pathways, which then cause damage to both vasculature andorgans. Various neuroendocrine counter-regulatory systems are alsoactivated, often compounding the problem. Even with immediate andaggressive treatment, this can progress to multiple organ dysfunctionsyndrome and eventually death. Hence, there remains a need for improvedtechniques for diagnosis and treatment of patients with infectiousdiseases, blood-borne infections, sepsis, or systemic inflammatoryresponse syndrome.

SUMMARY

The present invention provides for an engineered molecular opsonin thatmay be used to bind biological pathogens or identify subclasses orspecific pathogen species for use in devices and systems for treatmentand diagnosis of patients with infectious diseases, blood-borneinfections or sepsis; or in the identification of water- or food-bornepathogens. An aspect of the invention provides for mannose-bindinglectin (MBL), which is an abundant natural serum protein that is part ofthe innate immune system. The ability of this protein lectin to bind tosurface molecules on virtually all classes of biopathogens (viruses,bacteria, fungi, protozoans) make engineered forms of MBL extremelyuseful in diagnosing and treating infectious diseases and sepsis.

An embodiment of the present invention provides for a recombinantopsonin comprising a carbohydrate recognition domain of an opsonin, asubstrate binding domain, and a flexible peptide domain that links therecognition domain to the solid surface binding domain. In aspects ofthe invention, the carbohydrate recognition domain is a lectin orfragment of a lectin. Alternatively, the carbohydrate recognition domainis a collectin or ficollin, or a portion or fragment of these. In aparticular aspect, the carbohydrate recognition domain (CRD) comprisesthe portion of MBL starting at the residue proline 81 at the N-terminalend of the lectin portion of the engineered opsonin. In anotherparticular aspect, the carbohydrate recognition domain comprises theportion of MBL starting at the residue glycine 111 at the N-terminal endof the lectin portion for the engineered opsonin.

In a particular aspect of the invention, the substrate binding domain ofthe recombinant opsonin comprises one or more cysteine residues thatallow chemical cross-linking to a solid substrate. The solid substratemay comprise a magnetic microbead (which may be coated with protein A),a microporous membrane, a hollow-fiber reactor, or any other bloodfiltration membrane or flow device. In other aspects, the substrate canbe the surface of cells, such as immune cells (e.g., macrophages), thesurfaces of cells that line the tissues or organs of the immune system(e.g., lymph nodes or spleen), or the surface of the extracellularmatrix of tissues or organs of the immune system.

In another aspect of the invention, the flexible peptide domain maycomprise at least one Glycine+Serine segment and/or at least oneProline+Alanine+Serine segment. In another aspect of the presentinvention, the flexible linker is a Fc portion of immunoglobulin, suchas Fcγ. Fusion of human IgG1 Fc to the neck and CRD regions of MBLimproves the expression and purification and coupling to a substrate inan active form.

An embodiment of the invention provides for a method of collecting anopsonin-binding microorganism from a fluid comprising contacting thefluid with a recombinant opsonin conjugated to a solid surface; whereinthe recombinant opsonin consists of a carbohydrate recognition domain ofan opsonin, a solid substrate binding domain, and a flexible peptidedomain that links the recognition domain to the solid surface bindingdomain; allowing the opsonin-binding microorganism to bind to saidrecombinant opsonin-solid surface conjugate; and separating said fluidfrom said microorganism-bound recombinant opsonin-solid surfaceconjugate. The fluid may be a biological fluid, such as blood, obtainedfrom a subject. The fluid may then be returned to the subject.

Another embodiment of the invention provide a method of treating a bloodinfection in a subject comprising administering a recombinant opsonin tothe blood of the subject, wherein the recombinant opsonin consists of acarbohydrate recognition domain of an opsonin, a substrate bindingdomain, and a flexible peptide domain that links the recognition domainto the substrate binding domain, wherein the carbohydrate recognitiondomain binds an opsonin-binding microorganism, and wherein the substratebinding domain binds with a cell, tissue or organ of the immune system;allowing the recombinant opsonin to bind to the opsonin-bindingmicroorganism; and allowing the microorganism-bound recombinant opsoninto bind with a cell, tissue or organ of the immune system wherein themicroorganism is killed. The subject may be an animal or a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of mannose-binding lectin (MBL) engineered intosets of trimers (polymers) in an embodiment of the present invention.

FIGS. 2A and 2B are diagrams of an embodiment of the present inventionin which an artificial protein (FIG. 2A) comprising a stericallyunhindered N-terminus (optionally with a cysteine at or near theN-terminus), followed by a long, flexible peptide segment, then an MBLlectin domain at the C-terminus, is crosslinked to a solid substrate inthe example device in FIG. 2B.

FIG. 3 shows a diagram of an embodiment of the invention, Fc-MBL.81,both in cartoon and in model form based on the X ray crystallographymodels of Fc and of the neck and carbohydrate recognition domains (CRD)of MBL.

FIG. 4 is a scheme of a vector encoding Fc in an aspect of theinvention.

FIG. 5 shows the calcium-dependent binding of dynabead-MBL to C.albicans in which calcium maintains binding and EDTA destabilizesbinding.

FIG. 6 shows the binding of MBL-magnetic beads to different pathogens.Pathogens were bound by MBL-coated magnetic beads (control: beadswithout MBL), washed, and eluted onto culture plates.

FIG. 7 shows data from MBL-magnetic beads binding to microorganisms andovernight culture assay. The pathogens were bound by MBL-coated magneticbeads (control: beads without MBL), washed, and eluted onto cultureplates and incubated overnight.

FIGS. 8A and 8B demonstrate high levels of FcMBL expression fromtransient transfection. FIG. 8A is a western blot of a reduced gelloaded with unpurified supernatant of 293 cells transfected with pFUSEFcMBL.81 (and pFUSE Fc) probed with anti-hFc.

FIG. 8B shows Protein A-purified FcMBL.81.

FIG. 9 shows results of a depletion assay in which the FcMBL.81construct was as active as full-length MBL in binding C. albicans.

DETAILED DESCRIPTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

In the broadest sense, opsonins are proteins that bind to the surface ofa particle. In nature, opsonins act as binding enhancers for the processof phagocytosis, for example, by coating the negatively-chargedmolecules on a target pathogen's membrane. The present inventionprovides for an engineered molecular opsonin, such as mannose-bindinglectin (MBL), that may be used to bind biological pathogens or identifysubclasses or specific pathogen species for use in devices and systemsfor treatment and diagnosis of patients with infectious diseases,blood-borne infections or sepsis. Treatment may be carried out in vivoor ex vivo.

MBL is a serum lectin opsonin that binds to mannose, N-acetylglucosamine(NAG)-containing carbohydrates, and various other carbohydrates that arepresent on the surface of many microbial pathogens. MBL (also calledmannose- or mannan-binding protein, MBP) is a polymeric proteinassembled from three or more 32 kDa monomers. Each monomer has anN-terminal cysteine rich region, a collagen-like gly-X-Y region, a neckregion and a carbohydrate recognition domain. The assembly of the highermolecular weight (MW) polymers begins with formation of trimers of the32 kDa monomer; these trimers then self-assembly into higher MW polymersof three to six sets of trimers. See FIG. 1.

MBL is a key component in opsonization of microbial pathogens and in theactivation of complement (via the lectin pathway) and coagulation.Opsonization is the binding of proteins to target cells and thetargeting these cells for uptake and destruction by phagocytic cells,such as macrophages and neutrophils. This opsonization appears to bemediated by the small, cysteine-rich N-terminal domain of MBL as well asC3b deposited on the target cell surface by MBL-mediated lectincomplement pathway activation.

In the activation of complement via the lectin pathway, the microbe andspecialized proteins, i.e., MASP-1 (Mannan-binding lectin AssociatedSerine Protease) (Matsushita & Fujita, 176 J. Exp. Med. 1497 (1992)),and MASP-2 (Thiel et al., 386 Nat. 506 (1997)), interact with bound MBLand activate complement in the absence of antibody. The higher molecularweight MBL complexes (5 to 6 repeats of the functional MBL trimer) arepotent activators of complement via this lectin pathway, in which MASP 2appears to activate complement, and MASP 1 activates coagulation. Thesmaller complexes (three to four repeats of the MBL trimer unit) are themost potent activators of coagulation. Krarup et al., 2 PLoS One e623(2007).

In certain human populations, there is a high allele frequency ofmutations in MBL in the collagen helix, at codons 52, 54, and 57. Garredet al., 7 Genes Immun. 85 (2006). These mutations prevent the formationof the higher molecular weight MBL forms and suppress complementactivation. In these cases, MBL still functions as an opsonin andstimulates coagulation, but without activating complement. There is alsosome evidence for heterozygote advantage with respect to sepsis, in thatheterozygotes have the best survival, homozygous “wild-type” secondbest, and homozygous “mutant” have the worst survival. See Sprong etal., 49 Clin. Infect Dis. 1380 (2009). In addition, homozygous mutantneonates are particularly susceptible to infection before the acquiredimmune system begins to function.

There has been much debate on the usefulness of MBL as a recombinanttherapeutic protein for treatment of infectious diseases. Intact MBL hasbeen used in Phase 1 and Phase 2 clinical trials, both as a recombinantprotein and when purified from human blood donations. In fact,plasma-derived MBL has been used as a therapeutic in Phase 1 and PhaseII trials of MBL deficient, pediatric patients with chemotherapy inducedneutropenia. Frakking et al., 45 Eur. J. Cancer 50 (2009). Commercialefforts to develop MBL have foundered because of difficulties in bothproducing the recombinant protein and establishing efficacy. As usedherein, treatment or treating a subject can refer to medical careprovided to manage, improve, or relieve disease, illness, or symptomsthereof.

The present invention provides for engineered opsonins, e.g., engineeredMBL or MBL polymers, for use in devices and systems for pathogendetection and clearance. FIG. 5 shows the calcium-dependent binding ofMBL-conjugated magnetic microbeads to the yeast C. albicans. FIGS. 6 and7 compare the MBL-magnetic bead binding between several differencepathogens, including the gram positive bacterium, S. aureus; gramnegative bacteria, Klebsiella and E. coli; and yeast, C. albicans.Recent work has demonstrated the feasibility of using combinedmicromagnetic and microfluidic techniques to clear living pathogens fromflowing fluids, such as biological fluids, such as blood. Xia et al., 8Biomed. Dev. Biomed. Microdev. 299 (2006); Yung et al., Lab on a ChipDOI: 10.1039/b816986a (2009). In these microdevices (magnetic microbeadsthat are coated with molecules that bind specifically to surface markerson pathogen cells), are allowed to bind to these cells in whole humanblood, and then are pulled free from blood flowing through microfluidicchannels using an applied magnetic field gradient. See WO/2008/130618;WO/2007/044642.

Among other uses, these devices have great promise to rapidly clearblood of septic patients of toxin-producing pathogens, and hence greatlyincrease response to conventional antibiotic therapies. The ability torapidly (within minutes) bind, detect and isolate living pathogenscirculating in blood, or present within other biological fluids, using apotentially inexpensive and easy-to-use microdevice also circumvents themajor limitations of current pathogen detection and sensitivity testingassays that require multiple days of microbial culture in hospital orcommercial laboratories.

Biological fluids that may by used in the present invention include, forexample, blood, cerebrospinal fluid, joint fluid, urine, semen, saliva,tears, and fluids collected by insertion of a needle. Additionally,fluids may be collected from food or water samples for rapid, generalcontamination assays according to the present invention: such fluid canbe collected and analyzed for natural microbial contamination or forpossible “bio-terrorism” contamination.

Further, the current effectiveness of these methods harnesses priorknowledge of the specific pathogen that one desires to clear from theblood, because a specific ligand for that pathogen (e.g., specificantibody) is placed on the magnetic microbeads prior to using the bloodcleansing device. Thus, the present invention bolsters the currentapproaches by providing engineered generic binding molecules thatfunction like biological opsonins and bind to specific, many or all,types of microbial pathogens as the application requires. In thisregard, the present invention has therapeutic applications.

Another need addressed herein is the development of specialized pathogenclass-specific opsonins that bind, for example, all types of fungi orall gram negative bacteria or all or specific gram positive bacteria orall viruses or all protozoans, as this knowledge could quickly advisephysicians in their choice of anti-microbial therapies before completecharacterization of species type of antibiotic sensitivity is identifiedwith conventional methods that often take many days to complete.

In addition, with the use of genetic engineering, and directed evolutionand selection strategies, modified versions of natural opsonins can beengineered, such as MBL, that bind to pathogens in a species-specificmanner. Finally, binding that is specific for pathogen sensitivity todifferent antibiotics or antimicrobial therapeutics can be accomplishedusing appropriate selection strategies. Hence, this invention providesfor development of engineered opsonins that provide these high valueproperties.

MBL is an excellent choice for use as a generic opsonin for the purposesdescribed herein; however, the intact molecule is not typically used inthe presence of whole blood because it has multiple functional domainsthat promote blood coagulation that may interfere with diagnostic andtherapeutic microdevice function. This characteristic of MBL can beseparated from its pathogen binding function as provided herein. Morespecifically, MBL contains four parts, from N- to C-terminus: a smallN-terminal domain of essentially unknown function that may be involvedin macrophage binding and/or MASP binding; a collagen segment that mayalso be involved in MASP binding and higher-order oligomerization; analpha-helical “neck” segment that is sufficient for trimerization; andthe CRD lectin domain at the C-terminus that mediates direct pathogenbinding. The lectin domain is useful for the application at hand, andthe other domains may be present or deleted depending on the needs ofthe user, and can be determined by routine testing. Additionally, thelectin activity is calcium-dependent, so bound microbes could bereleased by a chelating agent for diagnostic purposes.

One embodiment of an engineered configuration of MBL, useful as ageneric opsonin for diagnostic and therapeutic applications, comprisesthe lectin domain of MBL. For example, Glycine 111 (as defined in theResearch Collaboratory for Structural Bioinformatics (RCSB), ProteinData Bank structure file 1HUP) is a convenient N-terminal point at whichto begin the lectin portion of the engineered opsonin. Because thebinding of MBL to a given monomeric sugar is weak, the MBL may beattached to the solid matrix in a flexible manner so that the proteinson the surface can move and adjust to the shape of the microbe. Forexample, a flexible peptide, such as one or more Glycine+Serine segmentor one or more Proline+Alanine+Serine segment, or other peptidelinker(s) known in the art, may be placed at the MBL N-terminus, as inFIG. 2A, because these segments tend to not form folded structures.

Another embodiment of an engineered configuration of MBL, useful as ageneric opsonin for diagnosis and therapeutic applications, comprisesthe neck and lectin domains of MBL. Proline 81 (as defined, for example,in the Research Collaboratory for Structural Bioinformatics, ProteinData Bank (RCSB PDB) structural file 1HUP) is a convenient N-terminalpoint at which to begin the lectin sequence for this engineered opsoninconstruct. This portion of MBL is fused downstream (C-terminal) to Fcportion of human IgG (Fcγ). The Fc portion may include the CH2-CH3interface of the IgG Fc domain, which contains the binding sites for anumber of Fc receptors including Staphylococcal protein A. In use, theFc portion dimerizes and strengthens the avidity affinity of the bindingby MBL lectins to monomeric sugars. Additionally, when used as adiagnostic reagent, the n-linked glycosylation of the recombinantopsonin can be removed. For example, in Fc MBL.81 the glycosylation canbe removed by changing the amino acid at residue 297 from asparagine toaspartic acid (N297D) in the Kabat system of numbering amino acids inantibodies, this corresponds to amino acid 82 in this particular Fcconstruct. Glycosylated Fc maintains the correct orientation for Fcmediated antibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-mediated cytotoxicity (CDC).

The engineered Fc MBL opsonin could be used in the activation of Fcreceptor-mediated uptake of Fc MBL opsonized Mycobacterium tuberculosis,bypassing mannose receptor mediated uptake of M. tuberculosis. Recentpublications (Kang et al., 202 J. Exp. Med. 987 (2005)), suggest thatlipoarabinomannan (ManLaM) on the cells surface of M. tuberculosisengage macrophage mannose receptor (MMR) during the phagocytic process.This directs M. tuberculosis to its initial phagosomal niche andinhibits phagosome-lysosome (P-L) fusion, thereby enhancing survival inhuman macrophages. Interestingly, inhibition of P-L fusion did not occurwith entry via Fcγ receptors. In one embodiment, uptake by Fc recetorendocytosis routes the bacterium, e.g., M. tuberculosis, to differentintracellular vesicles.

The configuration of the engineered opsonin of the present inventionalso aids attachment of the fusion protein to a substrate, such as asolid surface of a magnetic microbead or a microporous membrane, using achemical cross-linker that is specific for the amino group at theN-terminus, or to a free cysteine residue that has been engineered to benear the N-terminus of the protein, as in FIG. 2B. (Lysine is analternative to cysteine, optionally following removal of the rest of thelysine residues in the protein).

In some embodiments, the substrate to which the opsonin binds is aliving cell or extracellular matrix of a tissue or organ. For example,the substrate may be the surface of a cell, tissue or organ associatedwith the immune response. For example, the cell may be a phagocyte(macrophage, neutrophil, and dendritic cell), mast cell, eosinophil,basophil, and/or natural killer cell. The cell may be the cell oftissues or organs of the immune system, such as spleen, lymph nodes,lymphatic vessels, tonsils, thymus, bone marrow, Peyer's patches,connective tissues, mucous membranes, the reticuloendothelial system,etc. The surface to which the opsonin binds may also be theextracellular matrix of one or more of these tissues or organs.

In some embodiments, the solid substrate may comprise magnetic beads orother structured materials, which then pull microbes out from fluids,including biological fluids such as blood, and concentrate and collectthe microbes, including living microbes. This approach is advantageousbecause the beads can then be examined for the presence of the microbe,or be used to transfer the collected microbes to conventional pathogenculture and sensitivity testing assays. In other words, the engineeredopsonin may be used in diagnostics as a means of collecting potentialpathogens for identification; not only in the diagnosis of disease, butin the identification of water- or food-borne pathogens, particulates orother contaminants. Alternatively, the solid substrate may comprise ahollow-fiber reactor or any other blood filtration membrane or flowdevice (e.g., a simple dialysis tube) or other resins, fibers, or sheetsto selective bind and sequester the biological pathogens.

The magnetic beads can be of any shape, including but not limited tospherical, rod, elliptical, cylindrical, disc, and the like. In someembodiments, magnetic beads having a true spherical shape and definedsurface chemistry are used to minimize chemical agglutination andnon-specific binding. As used herein, the term “magnetic beads” refersto a nano- or micro-scale particle that is attracted or repelled by amagnetic field gradient or has a non-zero magnetic susceptibility. Themagnetic bead can be paramagnetic or super-paramagnetic. In someembodiments, magnetic beads are super-paramagnetic. Magnetic beads arealso referred to as magnetic particles herein. In some embodiments,magnetic beads having a polymer shell are used to protect the pathogenfrom exposure to iron. For example, polymer-coated magnetic beads can beused to protect pathogens from exposure to iron.

The magnetic beads can range in size from 1 nm to 1 mm. For example,magnetic beads are about 250 nm to about 250 μm in size. In someembodiments, magnetic bead is 0.1 μm to 100 μm in size. In someembodiments, magnetic bead is 0.1 μm to 50 μm in size. In someembodiments, magnetic bead is 0.1 μm to 10 μm in size. In someembodiments, the magnetic bead is a magnetic nano-particle or magneticmicro-particle. Magnetic nanoparticles are a class of nanoparticle whichcan be manipulated using magnetic field or magnetic field gradient. Suchparticles commonly consist of magnetic elements such as iron, nickel andcobalt and their chemical compounds. Magnetic nano-particles arewell-known and methods for their preparation have been described in theart. See, e.g., U.S. Pat. Nos. 6,878,445; 5,543,158; 5,578,325;6,676,729; 6,045,925; and 7,462,446; and U.S. Patent Publications No.2005/0025971; No. 2005/0200438; No. 2005/0201941; No. 2005/0271745; No.2006/0228551; No. 2006/0233712; No. 2007/01666232; and No. 2007/0264199.

Magnetic beads are easily and widely available commercially, with orwithout functional groups capable of binding to affinity molecules.Suitable magnetic beads are commercially available such as from DynalInc. (Lake Success, N.Y.); PerSeptive Diagnostics, Inc. (Cambridge,Mass.); Invitrogen Corp. (Carlsbad, Calif.); Cortex Biochem Inc. (SanLeandro, Calif.); and Bangs Laboratories (Fishers, Ind.). In particularembodiments, magnetic particles are MyOne™ Dynabeads® magnetic beads(Dynal Inc.).

The solid substrate can be fabricated from or coated with abiocompatible material. As used herein, the term “biocompatiblematerial” refers to any material that does not deteriorate appreciablyand does not induce a significant immune response or deleterious tissuereaction, e.g., toxic reaction or significant irritation, over time whenimplanted into or placed adjacent to the biological tissue of a subject,or induce blood clotting or coagulation when it comes in contact withblood. Suitable biocompatible materials include, for example,derivatives and copolymers of a polyimides, poly(ethylene glycol),polyvinyl alcohol, polyethyleneimine, and polyvinylamine, polyacrylates,polyamides, polyesters, polycarbonates, and polystyrenes.

In some embodiments, the solid substrate is fabricated or coated with amaterial selected from the group consisting of polydimethylsiloxane,polyimide, polyethylene terephthalate, polymethylmethacrylate,polyurethane, polyvinylchloride, polystyrene polysulfone, polycarbonate,polymethylpentene, polypropylene, a polyvinylidine fluoride,polysilicon, polytetrafluoroethylene, polysulfone, acrylonitrilebutadiene styrene, polyacrylonitrile, polybutadiene, poly(butyleneterephthalate), poly(ether sulfone), poly(ether ether ketones),poly(ethylene glycol), styrene-acrylonitrile resin, poly(trimethyleneterephthalate), polyvinyl butyral, polyvinylidenedifluoride, poly(vinylpyrrolidone), and any combination thereof.

In an aspect of the invention, the recombinant opsonins described hereincan be conjugated with the solid substrate by methods well known in theart for conjugating peptides with other molecules. For example,Hermanson, BIOCONJUGATE TECHNIQUES (2nd Ed., Academic Press (2008)) andNiemeyr, Bioconjugation Protocols: Strategies & Methods, in METHODS INMOLECULAR BIOLOGY (Humana Press, 2004), provide a number of methods andtechniques for conjugating peptides to other molecules. de Graaf, etal., 20 Biocojugate Chem. 1281 (2009), provides a review ofsite-specific introduction of non-natural amino acids into peptides forconjugation.

Alternatively, the surface of the solid substrate can be functionalizedto include binding molecules that bind selectively with the recombinantopsonin. These binding molecules are also referred to as affinitymolecules herein. The binding molecule can be bound covalently ornon-covalently on the surface of the solid substrate. As used herein,the term “binding molecule” or “affinity molecule” refers to anymolecule that is capable of specifically binding a recombinant opsonindescribed herein. Representative examples of affinity molecules include,but are not limited to, antibodies, antigens, lectins, proteins,peptides, nucleic acids (DNA, RNA, PNA and nucleic acids that aremixtures thereof or that include nucleotide derivatives or analogs);receptor molecules, such as the insulin receptor; ligands for receptors(e.g., insulin for the insulin receptor); and biological, chemical orother molecules that have affinity for another molecule, such as biotinand avidin. The binding molecules need not comprise an entire naturallyoccurring molecule but may consist of only a portion, fragment orsubunit of a naturally or non-naturally occurring molecule, as forexample the Fab fragment of an antibody. The binding molecule mayfurther comprise a marker that can be detected.

The binding molecule can be conjugated to surface of the solid substrateusing any of a variety of methods known to those of skill in the art.The binding molecule can be coupled or conjugated to surface of thesolid substrate covalently or non-covalently. Covalent immobilizationmay be accomplished through, for example, silane coupling. See, e.g.,Weetall, 15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 Meths. Enzymol.134 (1976). The covalent linkage between the binding molecule and thesurface can also be mediated by a linker. The non-covalent linkagebetween the affinity molecule and the surface can be based on ionicinteractions, van der Waals interactions, dipole-dipole interactions,hydrogen bonds, electrostatic interactions, and/or shape recognitioninteractions.

As used herein, the term “linker” means a molecular moiety that connectstwo parts of a composition. Peptide linkers may affect folding of agiven fusion protein, and may also react/bind with other proteins, andthese properties can be screened for by known techniques. Examplelinkers, in addition to those described herein, include is a string ofhistidine residues, e.g., His6; sequences made up of Ala and Pro,varying the number of Ala-Pro pairs to modulate the flexibility of thelinker; and sequences made up of charged amino acid residues e.g.,mixing Glu and Lys. Flexibility can be controlled by the types andnumbers of residues in the linker. See, e.g., Perham, 30 Biochem. 8501(1991); Wriggers et al., 80 Biopolymers 736 (2005). Chemical linkers maycomprise a direct bond or an atom such as oxygen or sulfur, a unit suchas NH, C(O), C(O)NH, SO, SO₂, SO₂NH, or a chain of atoms, such assubstituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedC₂-C₆ alkenyl, substituted or unsubstituted C₂-C₆ alkynyl, substitutedor unsubstituted C₆-C₁₂ aryl, substituted or unsubstituted C₅-C₁₂heteroaryl, substituted or unsubstituted C₅-C₁₂ heterocyclyl,substituted or unsubstituted C₃-C₁₂ cycloalkyl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, NH, orC(O).

Nucleic acid based binding molecules include aptamers. As used herein,the term “aptamer” means a single-stranded, partially single-stranded,partially double-stranded or double-stranded nucleotide sequence capableof specifically recognizing a selected non-oligonucleotide molecule orgroup of molecules by a mechanism other than Watson-Crick base pairingor triplex formation. Aptamers can include, without limitation, definedsequence segments and sequences comprising nucleotides, ribonucleotides,deoxyribonucleotides, nucleotide analogs, modified nucleotides andnucleotides comprising backbone modifications, branchpoints andnonnucleotide residues, groups or bridges. Methods for selectingaptamers for binding to a molecule are widely known in the art andeasily accessible to one of ordinary skill in the art.

The recombinant opsonin can be conjugated with surface of the solidsubstrate by an affinity binding pair. The term “affinity binding pair”or “binding pair” refers to first and second molecules that specificallybind to each other. One member of the binding pair is conjugated withthe solid substrate while the second member is conjugated with therecombinant opsonin. As used herein, the term “specific binding” refersto binding of the first member of the binding pair to the second memberof the binding pair with greater affinity and specificity than to othermolecules.

Exemplary binding pairs include any haptenic or antigenic compound incombination with a corresponding antibody or binding portion or fragmentthereof (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulinand goat antimouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin), hormone (e.g., thyroxine andcortisol-hormone binding protein), receptor-receptor agonist,receptor-receptor antagonist (e.g., acetylcholine receptor-acetylcholineor an analog thereof), IgG-protein A, lectin-carbohydrate, enzyme-enzymecofactor, enzyme-enzyme inhibitor, and complementary oligonucleoitdepairs capable of forming nucleic acid duplexes), and the like. Thebinding pair can also include a first molecule that is negativelycharged and a second molecule that is positively charged.

One example of using binding pair conjugation is the biotin-sandwichmethod. See, e.g., Davis et al., 103 PNAS 8155 (2006). The two moleculesto be conjugated together are biotinylated and then conjugated togetherusing tetravalent streptavidin as a linker. A peptide can be coupled tothe 15-amino acid sequence of an acceptor peptide for biotinylation(referred to as AP; Chen et al., 2 Nat. Methods 99 (2005)). The acceptorpeptide sequence allows site-specific biotinylation by the E. colienzyme biotin ligase (BirA; Id.). A recombinant opsonin can be similarlybiotinylated for conjugation with a solid substrate. Many commercialkits are also available for biotinylating proteins. Another example forconjugation to a solid surface would be to use PLP-mediatedbioconjugation. See, e.g., Witus et al., 132 JACS 16812 (2010). In thisexample, an AKT sequence on the Fc N terminal allows conjugation to thesolid surface and orientation of the lectin binding domain in theoptimal orientation pointing away from the solid surface.

It should be noted that the affinity of a single lectin domain for asugar is low, and binding is normally driven by avidity andmultivalency. In the case of the present devices, the multimerizationdomains are deleted from the protein, and multivalency of the protein iseffectively produced by attachment to a solid substrate (e.g., a bead)at high density, which density can be varied to provide optimalfunctionality.

Further regarding the MBL, its binding characteristics can bemanipulated by directed evolution for altered binding specificity. MBLmay be modified so that it binds to a more limited set of sugars orother molecular features, with the result that the modified MBL willbind to a more limited set of microbes to provide a capability forpathogen class identification (e.g., one of virus, bacteria, fungi, orprotozoan), subclass typing (e.g., gram negative or gram positivebacteria) or specific species determination. Numerous strategies areavailable in the art.

For example, a straightforward directed evolution strategy visuallyexamines an atomic structure of MBL complexed with a sugar, and thenmutates appropriate amino acids that make contact in a sugar-specificmanner, so that distinctive contacts are lost or particular types ofsteric hindrance are created. The three dimensional structure of rat MBLhas been solved in a complex with a high-mannose oligosaccharide andwith N-acetylglucosamine, a methylated fucose, and so on. His189Val andIle207Val are examples of substitutions that modifications alterspecificity.

In another strategy of directed evolution, the protein is subjected torandom mutagenesis and the resulting proteins are screened for desiredqualities. This is a particularly useful technology for affinitymaturation of phage display antibodies, where the antibody complementarydetermining regions (CDRs) are mutated by saturation mutagenesis andsuccessful variants of the six CDRs are shuffled together to form thehighest affinity antibodies.

The directed evolution paradigm can be applied to MBL in order to selectMBL variants with specific binding to yeast, gram-positive bacteria,gram-negative, coagulase negative, aerobic bacteria, etc. For this towork, however, the pattern and nature of the target sugars or relatedsurface features on these target organisms may have to differ betweenthe classes or species.

MBL is known to bind strongly to mannose and N-acetylglucosamine sugarson fungi, gram-positive, and gram-negative bacteria. For example, MBLbinds strongly to Candida spp., Aspergillus fumigatus, Staphylococcusaureus, and β hemolytic group A streptococci. MBL has intermediateaffinity to Escherichia coli, Klebsiella spp., and Haemophilusinfluenzae type b. MBL binds weakly to β hemolytic group B streptococci,Streptococcus pneumoniae, and Staphylococcus epidermidis. Neth et al.,68 Infect. & Immun. 688 (2000). The capsular polysaccharide of Neisseriameningitides serogroup B, H. influenzae type b and Cryptococcusneoformans are thought to decrease MBL binding, as does bacterialendotoxin. Id.; Van Emmerik et al., 97 Clin. Exp. Immunol. 411 (1994);Schelenz et al., 63 Infect. Immun. 3360 (1995).

Others have reported that MBL facilitates opsonophagocytosis of yeastsbut not of bacteria, despite MBL binding: MBL (Lectin) pathway ofcomplement was critical for the opsonophagocytosis of yeast, but theclassical complement pathway was critical for opsonophagocytosis ofbacteria. Brouwer et al., 180 J. Immunol. 4124 (2008). It was notreported that MBL bound to the bacterial species tested, however, onlythat MBL binding did not promote significant complement activation andopsonophagocytosis.

Derivatives of MBL with a particular specificity can be isolated by thefollowing approach, which is a standard phage display strategy: First,express a set of MBL variants from a phagemid vector; then bind thislibrary to a target of interest (e.g., E. coli) and perform one or tworounds of selection; and then perform a round of negative selectionagainst a related target (e.g., Candida), taking those phagemids thatfail to bind. These cycles of positive and negative selection are thenrepeated until a population of phages that generally bind to the targetand do not bind to the non-target is generated. This method may beapplied to any pair of microbial strains against which differentialbinding is desired, such as bacteria that are resistant and sensitive toa given antibiotic. This positive/negative enrichment strategy may alsobe used with an antibody-phage display library, which is an even morestandard way to isolate such specific binders.

MBL belongs to the class of collectins in the C-type (calcium-dependent)lectin superfamily, other members of which, such as surfactant proteinA, surfactant protein D, CL-L1 and CL-P1, may be useful in the presentinvention. Other possible opsonins include ficollins (Thiel et al.,1997), which also activate the lectin pathway of complement and bindMASP proteins. These proteins are related to MBL but have a different,more limited specificity. In the context of the diagnostic devicedescribed herein, one option is to simply use the lectin domain of aficollin that corresponds to the lectin domain of MBL described above.Another approach is to use ‘shuffling’ of segments or individual aminoacids between MBL and one or more Ficollins to create hybrid moleculesthat may have hybrid specificities. The directed evolution and selectionapproach described above also could potentially be used to generatehuman antibody fragments or peptides that provide the class, subclassand species specificity described above.

The present invention may be defined in any of the following numberedparagraphs:

1. A recombinant opsonin comprising: a carbohydrate recognition domainof an opsonin; a substrate binding domain; and a peptide domain thatlinks the recognition domain to the substrate binding domain.

2. The recombinant opsonin of paragraph 1, wherein said carbohydraterecognition domain is a collectin or ficollin or derived from acollectin or ficollin.

3. The recombinant opsonin of paragraph 1, wherein said carbohydraterecognition domain is a lectin or a portion or a fragment of a lectin.

4. The recombinant opsonin of paragraph 3, wherein said lectin ismannose-binding lectin (MBL).

5. The recombinant opsonin of paragraph 4, wherein the lectin consistsof amino acid residues 81 (proline) to 228 (isoleucine) of MBL (SEQ IDNO:2).

6. The recombinant opsonin of any of the foregoing paragraphs, whereinsaid substrate binding domain comprises at least one cysteine residuethat allows chemical cross-linking to a solid substrate.

7. The recombinant opsonin of any of the foregoing paragraphs, whereinthe flexible peptide comprises a Glycine+Serine segment or aProline+Alanine+Serine segment.

8. The recombinant opsonin of the foregoing paragraphs, where theflexible peptide comprises a portion of immunoglobulin Fc.

9. The recombinant opsonin of paragraph 8, wherein the Fc portionincludes the CH2-CH3 interface of the IgG Fc domain.

10. The recombinant opsonin of any of the foregoing paragraph, whereinthe substrate is a magnetic microbead, a paramagnetic microbead, amicroporous membrane, a hollow-fiber reactor, or any other fluidfiltration membrane or flow device.

11. The recombinant opsonin of any of the foregoing paragraphs, whereinthe substrate is a living cell or extracellular matrix of a biologicaltissue or organ.

12. The recombinant opsonin of paragraph 11, wherein the substrate is aphagocyte.

13. A method of collecting an opsonin-binding microorganism from a fluidcomprising contacting the fluid with a recombinant opsonin conjugated toa solid surface; wherein the recombinant opsonin consists of acarbohydrate recognition domain of an opsonin, a solid substrate bindingdomain, and a flexible peptide domain that links the recognition domainto the solid surface binding domain; allowing the opsonin-bindingmicroorganism to bind to said recombinant opsonin-solid surfaceconjugate; and separating said fluid from said microorganism-boundrecombinant opsonin-solid surface conjugate.

14. The method of paragraph 13, wherein the solid surface is a magneticparticle, and the separating is achieved by applying magnetic force tothe fluid after the opsonin-binding microorganism has bound to therecombinant opsonin-solid surface conjugate.

15. The method of paragraph 13, further comprising the step ofidentifying the microorganism.

16. The method of paragraph 13, wherein the fluid is a biological fluid.

17. The method of paragraph 16, wherein the biological fluid is selectedfrom the group consisting of blood, cerebrospinal fluid, joint fluid,urine, semen, saliva, tears, and fluids collected by needle, biopsy, oraspiration procedures.

18. The method of paragraph 17, wherein the biological fluid is blood.

19. The method of paragraph 18, further comprising the step of returningthe blood to its source.

20. The method of paragraph 19, wherein the source is a subject.

21. The method of paragraph 20, wherein the subject is suffering frominfection or sepsis.

22. The method of paragraph 13, wherein the fluid is derived from awater or a food sample.

23. The use of the recombinant opsonin of any of paragraphs 1 to 10 inthe identification of a pathogen.

24. The use of the recombinant opsonin of any of paragraphs 1 to 10 inthe diagnosis of disease.

25. The use of the recombinant opsonin of any of paragraphs 1 to 10 inthe identification of water or food contamination.

26. The use of the recombinant opsonin of any of paragraphs 1 to 12 inthe treatment of disease.

27. The use of the recombinant opsonin as in paragraph 26, furthercombined with additional treatment or therapy.

28. A method of treating a blood infection in a subject comprisingadministering a recombinant opsonin to the blood of the subject, whereinthe recombinant opsonin consists of a carbohydrate recognition domain ofan opsonin, a substrate binding domain, and a flexible peptide domainthat links the recognition domain to the substrate binding domain,wherein the carbohydrate recognition domain binds an opsonin-bindingmicroorganism, and wherein the substrate binding domain binds with acell, tissue or organ of the immune system; allowing the recombinantopsonin to bind to the opsonin-binding microorganism; and allowing themicroorganism-bound recombinant opsonin to bind with a cell, tissue ororgan of the immune system wherein the microorganism is killed.

29. The method of paragraph 28, wherein the subject is an animal.

30. The method of paragraph 28, wherein the subject is a human.

EXAMPLES Example 1. Construction and Expression of FcMBL.81

An embodiment of an engineered configuration of MBL, useful as a genericopsonin for diagnosis and therapeutic applications, was constructedusing the “neck” and “lectin” domains of MBL. Proline 81 (as defined inthe Research Collaboratory for Structural Bioinformatics, Protein DataBank structural file 1HUP) was selected as the N-terminal point at whichto begin the lectin sequence. This portion of the lectin molecule wasfused downstream (C-terminal) to Fc portion of human gamma 1 (Fcγ). Adiagram of the engineered opsonin construct is shown in FIG. 3. Aschematic of the Fc portion of a clone is shown in FIG. 4. The aminoacids for this construct include the following residues:

Fc Protein Sequence:

(SEQ ID NO: 1) 001epkssdktht cppcpapell ggpsvflfpp kpkdtlmisr tpevtcvvvd vshedpevkf 061nwyvdgvevh naktkpreeq ynstyrvvsv ltvlhqdwln gkeykckvsn kalpapiekt 121iskakgqpre pqvytlppsr deltknqvsl tclvkgfyps diavewesng qpennykttp 181pvldsdgsff lyskltvdks rwqqgnvfsc svmhealhnh ytqkslslsp gaMBL.81 Protein Sequence (this Includes the Coiled-Coil Neck Region andthe Carbohydrate Recognition Domains (CRD) of Human MBL):

(SEQ ID NO: 2) 81pdgdsslaas erkalqtema rikkwltfsl gkqvgnkffl tngeimtfek vkalcvkfqa 141svatprnaae ngaiqnlike eaflgitdek tegqfvdltg nrltytnwne gepnnagsde 201dcvlllkngq wndvpcstsh lavcefpiFc-MBL.81 Sequence:

(SEQ ID NO: 3) 001epkssdktht cppcpapell ggpsvflfpp kpkdtlmisr tpevtcvvvd vshedpevkf 061nwyvdgvevh naktkpreeq ynstyrvvsv ltvlhqdwln gkeykckvsn kalpapiekt 121iskakgqpre pqvytlppsr deltknqvsl tclvkgfyps diavewesng qpennykttp 181pvldsdgsff lyskltvdks rwqqgnvfsc svmhealhnh ytqkslslsp gapdgdssla 241aserkalqte marikkwltf slgkqvgnkf fltngeimtf ekvkalcvkf qasvatprna 301aengaiqnli keeaflgitd ekteggfvdl tgnrltytnw negepnnags dedcvlllkn 361gqwndvpcst shlavcefpi

Thus, the FcMBL.81 construct consists of a lectin having amino acidresidues 81 (proline) to 228 (isoleucine) of MBL, fused a portion ofFcγ. In use, the Fc portion dimerizes and adds avidity to the weakaffinity of the binding by MBL lectins to monomeric sugars. When FcMBL.81 is designed for use as a diagnostic reagent, the n-linkedglycosylation can be removed by changing the amino acid at 297 fromasparagine to aspartic acid (N297D), or amino acid 82 in the Fcconstruct. Glycosylated Fc is maintains the correct orientation for Fcmediated ADCC and CDC. Additionally, a cysteine residue can be clonedonto the engineered opsonin to allow binding to a solid substrate viachemical conjugation. The construction and expression of an engineeredopsonin, such as FcMBL, may be achieved by various techniques known inthe art, see, e.g., U.S. Pat. No. 5,541,087.

Expression of the construct in transiently transfected cells isdemonstrated in FIGS. 8A and 8B. The FcMBL.81 expressed about 35 mg/L.

Example 2. Comparison of Fc MBL.81 Construct with Full-Length MBL inBinding Yeast

Approximately 5.5 million Candida albicans yeast cells were inoculatedwith varying numbers of MBL beads coated with either wild-type,full-length MBL (hexamers of trimers) or Fc MBL.81. As depictedgraphically in FIG. 9, 18 million wild-type, full-length MBL or FcMBL.81 beads bound all 5.5 million fungal cells. This exampledemonstrates that Fc MBL.81 beads are as active as wild-type,full-length MBL beads in binding to C albicans.

We claim:
 1. A recombinant opsonin comprising an immunoglobulin Fc fusedto a carbohydrate recognition domain (CRD) of a mannose-binding lectin(MBL), wherein the CRD comprises amino acid residues 31 to 148 of SEQ IDNO: 2, and wherein the recombinant opsonin does not comprise thecysteine rich N-terminal domain of the MBL and does not comprise thecollagen-like segment of the MBL, wherein the recombinant opsonin iscovalently attached to a solid substrate.
 2. The recombinant opsonin ofclaim 1, wherein the solid substrate is selected from the groupconsisting of a magnetic microbead and a paramagnetic microbead.
 3. Therecombinant opsonin of claim 1, wherein the amino acid residues 31 to148 of SEQ ID NO: 2 are C-terminal of the immunoglobulin Fc.
 4. Therecombinant opsonin of claim 1, wherein the immunoglobulin Fc comprisesSEQ ID NO:
 1. 5. The recombinant opsonin of claim 1, wherein the solidsubstrate is selected from the group consisting of: a microporousmembrane, a hollow fiber, and a fluid filtration membrane.